Project Summary There remains an urgent need to develop medical countermeasures to treat acute and delayed radiation injuries that could result from a large-scale, radio-nuclear event. The unpredictable nature, as well as the complexity of the injuries from a nuclear blast makes this a challenging world health problem. If an event does occur, an immediate concern for survivors will be the development of opportunistic infections as radiation and injury damage the immune system. As there are limited treatment options for protecting the potentially high numbers of individuals that might be exposed to varying radiation, new and innovative strategies are necessary to advance the treatment of radiation injury. Cellular based therapy has shown benefit in numerous disease processes associated with injury and immune dysfunction. Mesenchymal stromal cells (MSCs) are ideal candidates for a cellular therapeutic as we have shown that they improve survival in murine sepsis by modulating the immune response and promoting resolution of inflammation. Pre-conditioning of MSCs may improve their efficacy in the oxidative inflammatory microenvironment into which they are administered after radiation injury. We have shown that the Toll-like receptor 9 agonist (CpG-ODN) may be an optimal candidate for pre-conditioning of MSCs. We found that a specific CpG-ODN sequence protected mice from post-injury infection and sepsis post-radiation. Thus, the overarching aim of this proposal is to develop MSCs and CpG pre-conditioned MSCs with the long- term goal of using CpG-MSCs as a cellular therapeutic to reduce the mortality and morbidity of radiation exposure injuries. We hypothesize that pre-conditioning MSCs by CpG-ODN stimulation will enhance their beneficial effects on anti-microbial immune function and hematopoietic system recovery to improve radiation injury and recovery. To test our hypothesis, we propose three aims. In Aim 1 we will systematically test and develop MSCs and CpG-ODN pre-conditioned MSCs as a treatment for radiation-induced injury and infection in a pre-clinical animal model looking at survival, bacterial counts, and immune cell recovery. Our preliminary data show that mice given CpG conditioned MSCs had a lower mortality rate following radiation and infection compared with untreated and MSC-treated mice. In Aim 2 we will explore the biological effects of CpG-ODN stimulation on MSC phenotype, function, homing to injury, response to oxidative stress, as well as their interaction with irradiated and non-irradiated immune cells. We will also use RNAseq to profile the CpG-ODN response of MSCs and identify mechanistic targets. In Aim 3 we will use data gained during the course of this project to identify mechanisms and pathways that mediate the beneficial activity of CpG-ODN conditioned MSCs. In this aim we will also determine whether treatment with MSCs and CpG MSCs impacts development of delayed effects of radiation. This research has significance, as knowledge gained from this study will provide pre-clinical data for translating CpG pre-conditioned MSCs as effective medical countermeasures for treating the variety of injuries that could occur following a radio-nuclear event.